Enhancement of the immune response using CD36-binding domain

ABSTRACT

The invention relates to reagents and methods for enhancing an immune response using CD36 binding region/antigen hybrid polypeptides or polynucleotides encoding the hybrid polypeptides.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Ser. No. 60/341,771filed Dec. 12, 2001, which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates to reagents and methods for enhancing animmune response using CD36 binding region/antigen hybrid polypeptides orpolynucleotides encoding the hybrid polypeptides.

BACKGROUND

[0003] Cells undergoing programmed cell death (i.e. apoptosis) arerecognized by phagocytes such as macrophages and immature dendriticcells (Albert, M. L., et al., “Immature dendritic cells phagocytoseapoptotic cells via alpha v beta 5 and CD36, and cross-present antigensto cytotoxic T lymphocytes,” J. Exp. Med. 188(7):1359 (1998)). Thisrecognition leads to the uptake and degradation of the dying cells.

[0004] Some of the molecular details of this recognition are now known.For example, recognition of and adherence to apoptotic cells byphagocytes occurs by several mechanisms including a CD36-dependentmechanism (Albert, M. L., et al., (1998)). CD36 is a cell surfaceglycoprotein that is expressed on dendritic cells, monocytes, andmacrophages (Platt, N., et al., Proc. Natl. Acad. Sci., 93: 12456(1996)). Furthermore, CD36 is a receptor for thrombospondin. (Asch etal. “Isolation of the thrombospondin membrane receptor,” J. Clin.Invest., 79:1054 (1987)).

[0005] Thrombospondins are a family of extracellular matrix adhesiveproteins. Thrombospondin 1 (TSP 1) inhibits angiogenesis and modulatesendothelial cell motility, adhesion, and cell growth. Thrombospondin 1has multiple functional domains, including a type I repeat which hashomology with properdin (Arch, A. S., et al., Biochem. Biophys. Res.Commun., 182(3):1208 (1992); Crombie, R., et al., J. Exp. Med., 187(1):25 (1998); Magnetto, S., et al., Cell Biochem. Funct, 16(3): 211 (1998);Carron, J. A., et al., Biochem. Biophys. Res. Commun. 270(3): 1124(2000); and Li, W., et al., J. Biol. Chem., 268:16179 (1993)). Withinthe type I repeat are two CSVTCG sequences that serve as binding sitesfor CD36. (Pearce, S. F. A., et al., “Recombinant GST/CD36 fusionproteins define a thrombospondin binding domain: evidence for a singlecalcium-dependent binding site on CD36,” J. Biol. Chem. 270: 2981(1995); and Asch, A. S., et al., “Thrombospondin sequence motif (CSVTCG)is responsible for CD36 binding,” Biochem. Biophys. Res. Commun. 182:1208 (1992)).

[0006] Engulfment of apoptotic bodies by phagocytes, including dendriticcells, is mediated by CD36 and results in cross-presentation of antigensto cytotoxic T-lymphocytes. (Albert M. L., et al. (1998)). Furthermore,human monocyte-derived macrophages phagocytose apoptotic neutrophils andeosinophils through a thrombospondin/CD36 dependent mechanism. (Stern etal., “Human monocyte-derived macrophage phagocytosis of eosinophilsundergoing apoptosis. Mediation by alpha v beta 3/CD36/thrombospondinrecognition mechanisms and lack phlogistic response,” Am. J. Pathol.149(3):911 (1996)). Therefore, CD36 and thrombospondin play an importantrole in the recognition and phagocytosis of apoptotic cells.

[0007] Classical vaccine technology has included the development of bothlive and inactivated vaccines. Live vaccines are typically attenuatednon-pathogenic versions of an infectious agent that are capable ofpriming an immune response directed against a pathogenic version of theinfectious agent. In recent years there have been advances in thedevelopment of live recombinant vaccines (e.g., recombinant poxviruses)in which foreign antigens of interest are expressed from a viral vector.Although there are numerous examples of live vaccines that are veryeffective (eg., vaccinia in the eradication of smallpox), there areinherent risks associated with live vectors. For example, it is possibleto contaminate live vaccines with harmful adventitious agents, sincelive vaccines cannot be subjected to harsh inactivation or purificationprocedures. Additionally, there is a possibility when using live vaccinevectors, of “runaway vaccination” causing systemic viremia inimmunocomprised recipients. Finally, live viral vectors elicit stronginflammatory and immunogenic responses against vector components thatlimit the utility of repeat administration.

[0008] Inactivated vaccines are comprised of killed whole pathogens, orsoluble proteins or protein subunits. While generally considered safe,the efficacy of inactivated vaccines at eliciting broad, long-lastingresponses is of concern for some vaccine preparations. In fact, mostinactivated vaccines fail to produce a significant CD8+ cellularresponse necessary for cytolytic immune activity. Recombinant proteinsare promising inactive vaccine or immunogenic composition candidatesbecause they can be produced at high yield and purity. However,recombinant proteins can be poorly immunogenic. Therefore, there is aneed for methods and compositions that enhance the immune response toinactivated vaccines, especially vaccines containing recombinantproteins.

[0009] Adjuvants have been used for many years to enhance the immuneresponse to antigens present in vaccines, including subunit or componentvaccines comprised of recombinant proteins. Currently, alum is the mostcommonly used adjuvant for human administration. However, although itsefficacy has been established, it is ineffective for certainvaccinations (e.g. influenza vaccination) and inconsistently elicits animmune response with certain immunogens. Therefore, there remains a needfor safe, effective, and easily manufactured compositions and methodsfor enhancing an immune response.

[0010] Recently, nucleic acid vectored vaccines (NAVAC) have beendeveloped. These vaccines involve direct inoculation of an organism withnucleic acid vectors containing inserts encoding antigens (i.e. geneticvaccination). It appears that both the method (e.g. intramuscularinjection, epidermal Gene Gun-mediated administration) and route ofinoculation (e.g. intramuscular, epidermal, mucosal) are important indetermining the efficacy of the immune response for NAVAC (Robinson etal., Vaccine 11:957 (1993); Ulmer et al., Vaccine 15:792 (1997); andShiver et al., Vaccine, 15:884 (1997)). This has led to inconsistentstimulation of an immune response using NAVAC. Therefore, there remainsa need for effective methods for using NAVAC to consistently elicit astrong immune response.

[0011] Despite our increasing understanding of the complex cellular andmolecular interactions involved in certain aspects of an immuneresponse, there remains a need for improved methods and compositions,such as vaccines, for enhancing an immune response. More specifically,there remains a need to develop methods and reagents that utilize ourgrowing understanding of antigen presenting cell-recognition molecules,such as CD36, and pathways that depend on these molecules, such asCD36/thrombospondin-dependent pathways.

SUMMARY OF THE INVENTION

[0012] The present invention provides improved methods for immunizing ahost against an antigen. In one embodiment, an isolated chimeric nucleicacid molecule encoding a chimeric polypeptide is provided. In apreferred embodiment, the chimeric nucleic acid molecule comprises anucleic acid sequence encoding at least one CD36 binding domain andanother nucleic acid sequence encoding at least one immunogenic aminoacid sequence (i.e., antigen). Following introduction of the nucleicacid molecule into a host, a chimeric polypeptide is expressed resultingin an immune response against the immunogenic amino acid sequence. Thus,in one embodiment, the present invention provides an isolated nucleicacid molecule encoding an immunogenic chimeric polypeptide, the nucleicacid molecule being suitable for administration to a host, either aloneor as a pharmaceutical composition. In another embodiment, the presentinvention provides an immunogenic chimeric polypeptide suitable foradministration to a host, either alone or as a pharmaceuticalcomposition. In yet another embodiment, a method of immunizing a hostusing the isolated nucleic acid molecule or the chimeric polypeptide isprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 illustrates a restriction map (FIG. 1A), the nucleotidesequence (FIG. 1B; see also GenBank Accession Number X14787) and theamino acid sequence (FIGS. 1B and 1C) of mature thrombospondin 1 (TSP1).The signal sequence is found at amino acids 1-31 and the connectingpeptide at amino acids 32-44; the CD36 binding domains are found atamino acids 504-511 and 447-452; and, the beta sheet region is found atamino acids 453-463.

[0014]FIG. 2 illustrates the structure (FIG. 2A) and nucleotide as wellas amino acid sequence (FIG. 2B) of the chimera pVITHROMBgp120FU. InFIG. 2B, The N-terminus of the thrombo=gp120 hybrid polypeptide beginsat the methionine residue encoded by the codon adjacent to the Pst Isite.

[0015]FIG. 3 shows V3 CTL activity following a single immunization withcontrol plasmid (open squares), plasmid gp120 (filled diamonds), plasmidthrombo=gp120 (filled triangles) or vP1008 (open squares).

[0016]FIG. 4 shows V3 CTL activity three weeks after a secondimmunization given three weeks after a first immunization, with controlplasmid (open squares), plasmid gp120 (filled diamonds), plasmidthrombo=gp120 (filled triangles) or vP1008 (open squares).

[0017]FIG. 5 shows anti-gp160 antibody production following immunizationduring weeks 0 and 3 with plasmid thrombo=gp120. Individual sera fromeach mouse was diluted 1:400 and assayed by kinetics ELISA (KELISA)using recombinant HIV MN/BRU envelope glycoprotein as antigen.

[0018]FIG. 6 shows V3 activity three weeks after a second immunizationwith control plasmid (open squares), plasmid gp120 (filled diamonds),plasmid thrombo=gp120 (filled triangles) or vP1008 (open squares).

[0019]FIG. 7 shows V3 (gp120) CTL activity at seven weeks after a secondimmunization with control plasmid (open squares), plasmid gp120 (filleddiamonds), plasmid thrombo=gp120 (filled triangles) or vP1008 (opensquares).

[0020]FIG. 8 shows anti-gp160 antibody responses to gp120 following twosuccessive immunizations with control plasmid (vical), plasmid gp120(vical-gp120), plasmid thrombo=gp120 or vP1008 after a firstimmunization (week 0) and a second immunization (week 3). KELISA resultsfor three mice from each group are graphed individually (open squares,closed triangles, and closed diamonds). Antibody responses werecalculated at the time of immunization, and at 3 weeks, 6 weeks, 8weeks, and 10 weeks after the first immunization.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention relates to a chimeric nucleic acid moleculecomprising a nucleic acid sequence encoding one or more CD36 bindingregions positioned in-frame to one or more nucleic acid sequencesencoding one or more immunogenic amino acid sequences (i.e., antigens).Such as nucleic acid molecule is referred to as a chimeric or hybridnucleic acid. The nucleic acid sequence(s) encoding the one or more CD36binding region(s) may be positioned upstream (5′) or downstream (3′)from the nucleic acid sequence(s) encoding the one or more antigens,provided a chimeric polypeptide comprising both the CD36 binding regionand antigenic region is expressed in cells transfected with the chimericnucleic acid molecule. Preferably, the nucleic acid sequence(s) encodingthe one or more CD36 binding region(s) is ligated upstream from thenucleic acid(s) encoding the one or more antigens. In preferredembodiments, the chimeric nucleic acid molecule is admixed in apharmaceutically acceptable carrier to form the immunogenic compositionsof the current invention. In other preferred embodiments, the chimericpolypeptide is admixed in a pharmaceutically acceptable carrier to formthe immunogenic compositions of the current invention. Recombinant DNAmethods used herein are generally those set forth in commonly usedreferences such as Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1989) and/or CurrentProtocols in Molecular Biology (Ausubel et al., Eds., Green publishersinc. and Wiley and Sons 1994). All references cited in this applicationare expressly incorporated by reference herein.

[0022] The term “CD36 binding region” means a polypeptide sequencecapable of binding to the cell surface glycoprotein CD36 in solid-phasebinding assays (see, for example, Crombie et al., J. Exp. Med., 187: 25(1998); and Pearce et al., J. Biol. Chem. 270: 2981 (1995)). Typically,the CD36 binding region includes sequences that do not bind CD36 in theabsence of other sequences within the CD36 binding region. Theboundaries of the CD36 binding region are typically defined byidentifying the amino or carboxy-most polypeptide subsegments within theCD36 binding region that are capable of binding CD36 or enhancingbinding to CD36 in solid-phase binding assays. In a preferredembodiment, the CD36 binding region of the present invention has theamino acid sequence of the CD36 binding region of TSP1 (SEQ ID NO.: 1;FIGS. 1B and 1C; GenBank Accession No. X14787). For the purposes ofpracticing the present invention, this region preferably comprises aminoacids 447-463 ligated with or without intervening amino acids to aminoacids 504-511 of TSP1.

[0023] Within the CD36 binding region are CD36 binding domains, whichare subsegments of the CD36 binding region that independently bind CD36in solid-phase binding assays. Binding of the CD36 binding region to thecell surface glycoprotein CD36 occurs through one or more of these CD36binding domains. The CD36 binding domain typically comprises the aminoacid sequence CSVTCG or sequences related thereto. A sequence related tothe sequence CSVTCG, or “consisting essentially of” CSVTCG is a sequencethat has at least 4 of the amino acids in CSVTCG, or comprisesconservative substitutions of at least 4 of the amino acids in CSVTCG,and retains the ability to bind CD36 in solid-phase binding assays.Representative CD36 binding domains are found at amino acids 447-452 andamino acids 504-511 of TSP1 (FIGS. 1B and 1C).

[0024] In one embodiment, the CD36 binding region contains at least twoCD36 binding domains. The domains are typically separated by spacerregions (or “spacer amino acids”) that preferably have a beta sheetconformation. Methods are known in the art for predicting whether apolypeptide region will have a beta sheet conformation based on theprimary sequence of the polypeptide. In addition, the conformation ofpolypeptide regions can be determined experimentally using well-knownmethods. The beta sheet spacers may be of any length provided that theresultant CD36 binding region enhances an immune response. It ispreferred that the spacers are approximately 11 amino acids in length.It is more preferred that the spacer be 11 amino acids in length. In amost preferred embodiment, the spacer is identical to amino acids453-463 of TSP1 or has the sequence DGVITRIRLCN (FIGS. 1B and 1C). In apreferred embodiment, the CD36 binding region contains two CD36 bindingdomains separated by a spacer region of about 11 amino acids.

[0025] The nucleic acid sequence(s) encoding the one or more antigensencodes an amino acid sequence that causes an immune response within ahost upon expression within the host. It is preferred that, followingexpression of an effective amount of chimeric nucleic acid oradministration of an effective amount the chimeric polypeptide, the hostis immunized against the antigen. An “effective amount” is that whichenhances an immune response as measured using assays known in the artincluding, for example, antibody assays, antigen specific cytotoxicityassays, and assays measuring the expression of cytokines. In preferredembodiments, the chimeric nucleic acid molecule or chimeric polypeptidefunctions as a vaccine. As is understood in the art, a vaccine is animmunogenic composition containing an antigen that, when administered toan animal, stimulates an immune response that at least partiallyprotects the animal from challenge by a cell or organism expressing theantigen. “Partially protects the animal” means that the immunogeniccomposition elicits an antibody-based or cytotoxic T lymphocyte responseagainst the antigen.

[0026] For example, where the antigen is a tumor antigen, the host ispreferably protected from the development of a tumor and/or the hostacquires the ability to eliminate an existing tumor from the body. Theterm “tumor antigen” as used herein includes both tumor associatedantigens (TAAs) and tumor specific antigens (TSAs), where a cancerouscell is the source of the antigen. A TAA is an antigen that is expressedon the surface of a tumor cell in higher amounts than is observed onnormal cells or an antigen that is expressed on normal cells duringfetal development. A TSA is an antigen that is unique to tumor cells andis not expressed on normal cells. The term tumor antigen includes TAAsor TSAs, antigenic fragments thereof, and modified versions that retaintheir antigenicity.

[0027] Suitable TAA or TSA may include wild-type or mutated antigenssuch as, for example, gp100 (Cox et al., Science, 264:716-719 (1994)),MART-1/Melan A (Kawakami et al., J. Exp. Med., 180:347-352 (1994)), gp75(TRP-1) (Wang et al., J. Exp. Med., 186:1131-1140 (1996)), tyrosinase(Wolfel et al., Eur. J. Immunol., 24:759-764 (1994)), NY-ESO-1 (WO99/18206) melanoma proteoglycan (Hellstrom et al., J. Immunol.,130:1467-1472 (1983)), antigens of MAGE family (i.e., MAGE-1, 2, 3, 4,6, and 12; Van der Bruggen et al., Science, 254:1643-1647 (1991)),antigens of BAGE family (Boel et al., Immunity, 2:167-175 (1995)),antigens of GAGE family (i.e., GAGE-1,2; Van den Eynde et al., J. Exp.Med., 182:689-698 (1995)), antigens of RAGE family (i.e., RAGE-1;Gaugler et at., Immunogenetics, 44:323-330 (1996)),N-acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med.,183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950(1995)), β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)),MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)),cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284(1995)), p21 ras (Fossum et at., Int. J. Cancer, 56:40-45 (1994)),BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald etal., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu(Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermal growthfactor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29:1-2(1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. CancerInst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087; 5,571,710;6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483);carcinoma-associated mutated mucins such as MUC-1 gene products (Jeromeet al., J. Immunol., 151:1654-1662 (1993)); EBNA gene products of EBV,for example, EBNA-1 gene product (Rickinson et al., Cancer Surveys,13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing etal., J. Immunol, 154:5934-5943 (1995)); prostate specific antigens (PSA)(Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membraneantigen (PSMA) (Israeli, et al., Cancer Res., 54:1807-1811 (1994));idiotypic epitopes or antigens, for example, immunoglobulin idiotypes orT cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787(1994)); KSA (U.S. Pat. No. 5,348,887), NY-ESO-1 (WO 98/14464), andNY-BR-1 (WO 01/47959). Other suitable tumor antigens are known in theart.

[0028] Similarly, where the antigen is derived from an infectious agentsuch as a bacterium, virus, parasite or fungus, the host is preferablyprotected from infection by an organism expressing the antigen and/orthe host acquires the ability to eliminate an existing infection. Forexample, where the antigen is the HIV gp120 protein or an immunogenicfragment derived therefrom, the host is preferably protected frominfection by HIV and/or the host acquires the ability to eliminate HIValready existing in the host.

[0029] The chimeric nucleic acid molecule may further include a nucleicacid sequence encoding an immunostimulatory molecule. Many suitableimmunostimulatory molecules are available to one of skill in the artincluding, for example, cytokines (e.g., interleukin-2 (IL-2),interleukin-12 (IL-12), and granulocyte-macrophage colony stimulatingfactor (GM-CSF)), co-stimulatory molecules (e.g., the B7 family ofmolecules such as B7.1 and B7.2), and/or other lymphokines that enhancethe immune response. In certain embodiments of the current invention,the immunogenic polypeptide composition of the current inventionincludes both a CD36 binding/immunogen chimeric polypeptide and animmunostimulatory molecule, in admixture with a pharmaceuticallyacceptable diluent or carrier.

[0030] It is preferred that the nucleic acid sequences encoding the CD36binding region and the antigen be operably linked so as to be expressedin a cell as a chimeric polypeptide. In one embodiment, the CD36 bindingregion(s) may be coupled to the amino terminus or carboxy terminus ofthe antigen coding region. Alternatively, the CD36 binding region may becoupled to a region within the antigen, provided that at least oneimmunologically effective epitope (i.e., an epitope capable of elicitingan immune response) and preferably all immunologically effectiveepitopes of the antigen are preserved. In a preferred embodiment, thefusion construct is constructed by ligating a polynucleotide encoding aCD36 binding region in frame to a polynucleotide encoding the antigenand expressing the ligated polynucleotide in a cell transfected with thepolynucleotide.

[0031] It is also possible to generate the chimeric polypeptide bylinking the CD36 binding region to the antigen indirectly through eithera covalent or non-covalent association. Preferably, the linkage betweenthe CD36 binding region and the antigen is covalent. Methods ofperforming such linkages are known in the art (see, for example,“Chemistry of Protein Conjugation and Crosslinking”. 1991, Shans Wong,CRC Press, Ann Arbor). Suitable crosslinking agents known in the artinclude, for example, the homobifunctional agents glutaraldehyde,dimethyladipimidate and bis(diazobenzidine) and the heterobifunctionalagents m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-mmaleimidobenzoyl-N-hydroxysuccinimide. Other suitable agents are knownin the art.

[0032] In one embodiment, indirect linkage of the CD36 binding regionmay be accomplished using a binding pair. For these embodiments, theCD36 binding region is coupled to a first binding pair member that iscapable of binding under physiological conditions to a second bindingpair member that is coupled to the antigen. Many suitable binding pairs,such as biotin and avidin, are known in the art. Preferably, the CD36binding region is coupled to the amino terminus of the antigen-codingregion.

[0033] In certain embodiments, the CD36 binding region/antigen chimericpolypeptides of the current invention are secreted from cells. It ispreferred that the polypeptides are expressed in an immature formcontaining a signal sequence, which is cleaved during the secretionprocess. The signal sequence may be derived from either a prokaryote ora eukaryote. For example, a prokaryotic signal sequence may be usedwhere eukaryotic post-translational modifications of the fusionpolypeptide are not necessary to elicit an enhanced immune responseagainst the antigen. For these situations, the chimeric polypeptides maybe expressed and secreted in vitro in a bacterial host cell.

[0034] In certain embodiments, the signal sequence comprises anendoplasmic reticulum (ER)-targeting sequence and a signal peptidase(SP) sequence. Inclusion of these sequences typically results insecretion of the CD36 binding region/antigen chimeric polypeptides fromcells transfected with a CD36 binding region/antigen chimericpolynucleotide of the current invention. In a preferred embodiment, theER/SP sequences comprise amino acid residues 1-44 of TSP1 as shown inSEQ ID NO.: 2 and FIGS. 1B and 1C. Other ER/SP sequences and methods foridentifying such sequences are known in the art (Goa et al., “Aids Res.and Human Retroviruses, 10: 1359 (1994)). In preferred embodiments, theER/SP sequence is positioned 5′ of the one or more nucleic acidsequences encoding a CD36 binding region and the one or more nucleicacid sequences encoding the antigen.

[0035] In another aspect, the present invention provides an isolatedform of the chimeric CD36 binding/immunogen polynucleotide of thepresent invention, wherein a nucleic acid sequence encoding a CD36binding region is ligated in frame to a nucleic acid sequence encodingan antigen. In one embodiment, the present invention provides a chimericpolynucleotide including a secretion signal sequence ligated upstream ofthe CD36 binding sequence which is ligated upstream of theantigen-coding sequence.

[0036] By “isolated” nucleic acid is intended a nucleic acid molecule,DNA or RNA, which has been removed from its native environment. IsolatedDNA molecules include recombinant DNA molecules maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules include in vivo, or invitro RNA transcripts of the DNA molecules of the present invention.Isolated nucleic acid molecules according to the present inventionfurther include such molecules produced synthetically. Recombinant DNAmolecules contained in a vector are also considered isolated for thepurposes of the present invention. The isolated nucleic acid moleculesand expression vectors of the present invention may be constructed usingstandard recombinant techniques widely available to one skilled in theart. Such techniques may be found in common molecular biology referencessuch as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), and PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calif.).

[0037] In preferred embodiments, the isolated nucleic acids of thecurrent invention include a transcriptional regulatory region that isoperatively-linked to the chimeric nucleic acid molecule. By“operatively linked” is meant transcription of the chimericpolynucleotide is affected by the activity of the transcriptionalregulatory region. Preferably, the transcriptional regulatory regiondrives high-level gene expression in the target cell. Thetranscriptional regulatory region may comprise, for example, a promoter,enhancer, silencer, repressor element, or combinations thereof. A widevariety of promoters can be utilized for the current invention. Suitabletranscriptional regulatory regions include, for example, the CMVpromoter (i.e., the CMV-E1 promoter shown in FIGS. 2A and 2B); the SV40late promoter; promoters from eukaryotic genes, such as theestrogen-inducible chicken ovalbumin gene, the interferon genes, thegluco-corticoid-inducible tyrosine aminotransferase gene, and thethymidine kinase gene; and the major early and late adenovirus genepromoters. Furthermore, as a large number of retroviruses are known thatinfect a wide range of eukaryotic host cells, the long terminal repeats(LTRs) frequently may also suffice as transcriptional regulatoryregions. It is also possible to operably link the chimericpolynucleotide to a tissue- or cell-specific transcriptional regulatoryregion to affect expression of the chimeric polynucleotide in certaincells or tissues. As such, when the isolated CD36 binding/antigenpolynucleotide is inserted into a cell, transcription of the chimericpolynucleotide is induced resulting in expression of the CD36binding/antigen chimeric polypeptide.

[0038] The chimeric polynucleotides are preferably administered as partof an expression vector. The vectors can be either RNA or DNA, eitherprokaryotic or eukaryotic, and are typically of viral or plasmid origin.As such, the chimeric polynucleotide is inserted into an expressionvector such that the polynucleotide is expressed when transformed into ahost cell.

[0039] Expression vectors of the present invention include any vectorsthat function (i.e., direct gene expression) in recombinant cells of thepresent invention, including in bacterial, fungal, parasite, insect,animal, and plant cells. Preferred expression vectors of the presentinvention can direct gene expression in animal cells, preferablymammalian, most preferably human cells.

[0040] There are many methods and vectors available for expressingpolynucleotides in cells. For instance, several types of mammalianexpression systems are known in the art. (See e.g., Sambrook et al.,“Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: ALaboratory Manual, 2nd ed. (1989)). Viral transduction methods maycomprise the use of a recombinant DNA or an RNA virus comprising anucleic acid sequence encoding the chimeric polypeptide to infect acell, resulting in expression of the chimeric polypeptide. Suitableviral vectors include, for example, adenovirus, pox viruses (i.e.,vaccinia or avipox), polio virus, and alphavirus, among others known inthe art.

[0041] Examples of suitable retroviral vectors include, but are notlimited to Moloney murine leukemia virus (MoMuLV), Harvey murine sarcomavirus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV andRous Sarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. Since recombinant retroviruses aredefective, they require assistance in order to produce infectious vectorparticles. This assistance can be provided, for example, by using helpercell lines that contain plasmids encoding all of the structural genes ofthe retrovirus under the control of regulatory sequences within the LTR.These plasmids are missing a nucleotide sequence which enables thepackaging mechanism to recognize an RNA transcript for encapsitation.Helper cell lines which have deletions of the packaging signal includebut are not limited to Ψ2, PA317 and PA12, for example.

[0042] In a preferred embodiment, the viral vector is a poxvirus such asvaccinia virus (Smith, et al., 1983, Gene, 25 (1): 21-8; Moss, 1992,Biotechnology, 20: 345-62; Moss, 1992, Curr. Top. Microbiol. Immunol.,158: 25-38; U.S. Pat. Nos. 5,364,773, 5,990,091, and 5,174,993). Incertain embodiments, a highly attenuated strain of vaccinia, designatedMVA, may be used as a vector (U.S. Pat. No. 5,185,146). A preferredvector is the NYVAC vector (U.S. Pat. Nos. 5,364,773 and 5,494,807). Inother preferred embodiments, the poxvirus vector is ALVAC (1) or ALVAC(2), both of which are derived from canarypox virus (see, for example,U.S. Pat. Nos. 5,833,975 and 5,990,091; Tartaglia, et al., J. Virol. 67:2370 (1993)). Fowlpox virus is another avipoxvirus that may also be usedin practicing the present invention (see, for example, U.S. Pat. No.5,766,599). Other suitable poxvirus vectors are known in the art.

[0043] In certain embodiments, the vector is a plasmid vector. Manyplasmid expression vectors are known in the art and could be used withthe current invention. In preferred embodiments, isolated nucleic acidsare directly administered to an animal, virtually any expression vectorthat is effective in animal cells can be used. Preferred vectors wherethe isolated nucleic acids of the current invention are intended forNAVAC applications.

[0044] Bacterial vectors may also be used with the current invention.These vectors include, for example, Shigella, Salmonella, Vibriocholerae, Lactobacillus, Bacille Calmette Guérin (BCG), andStreptococcus (See e.g., WO 88/6626; WO 90/0594; WO 91/13157; WO92/1796; and WO 92/21376). In these bacterial vector embodiments of thisinvention, a chimeric CD36 binding/immunogen polynucleotide of theinvention may be inserted into the bacterial genome, may remain in afree state, or may be carried on a plasmid (as described above). Othersuitable vectors include the E. coli expression vector pUR278 and theglutathione S-transferase (GST) vector pGEX.

[0045] Other delivery techniques including DNA-ligand complexes,adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄precipitation, gene gun techniques, electroporation, liposomes andlipofection (Mulligan, R., 1993, Science, 260 (5110): 926-32) have alsobeen demonstrated to be useful. Lipofection may be accomplished byencapsulating an isolated DNA molecule within a liposomal particle andcontacting the liposomal particle with the cell membrane of the targetcell. Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 μm canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form. Other suitable methods are available to one skilled in theart, and it is to be understood that the present invention may beaccomplished using any of the available methods of transfection.

[0046] The current invention further provides isolated CD36binding/immunogen chimeric polypeptides. The term “isolated” as usedherein refers to the removal of a polypeptide from its naturalenvironment and does not imply any specific level of purity of thepolypeptide. Many methods are known in the art that can be used toprepare the CD36 binding/immunogen chimeric polypeptides. For example,the fusion polypeptides may be prepared as recombinant fusionpolypeptides using the CD36 binding/immunogen expression polynucleotidesand recombinant cell lines. The CD36 binding/immunogen chimericpolypeptides may also be prepared by covalently linking the CD36 bindingregion to the antigen using chemical cross-linking methods andcross-linking agents well-known in the art as described above. (see, forexample, “Chemistry of Protein Conjugation and Crosslinking”. 1991,Shans Wong, CRC Press, Ann Arbor).

[0047] In certain embodiments, the isolated polypeptides of the currentinvention include additional purification fusion polypeptide segmentsthat assist in purification of the polypeptides. Suitable fusionsegments include, among others, metal binding domains (e.g., apoly-histidine segment), immunoglobulin binding domains (e.g., ProteinA; Protein G; T cell; B cell; Fc receptor or complement proteinantibody-binding domains), sugar binding domains (e.g., a maltosebinding domain), and/or a “tag” domain (e.g., at least a portion ofα-galactosidase, a strep tag peptide, a T7 tag peptide, a Flag peptide,or other domains that can be purified using compounds that bind to thedomain, such as monoclonal antibodies). Other suitable fusion segmentsare well known in the art.

[0048] In another aspect, the current invention includes recombinantcells and cell lines that express the CD36 binding/immunogen fusionpolypeptides of the current invention. The recombinant cells and celllines may be prokaryotic or preferably eukaryotic cells, that aretransformed with one or more CD36 binding/immunogen expression vector. Acell can be “transformed,” as the term is used in this specification,with a nucleic acid molecule, such as a recombinant expression vector,by any method by which a nucleic acid molecule can be introduced intothe cell. Transformation techniques include, but are not limited to,transfection, infection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. Transformation may be stable ortransient. A “cell line” refers to any immortalized recombinant cell ofthe present invention that is not a transgenic animal. Transformednucleic acid molecules of the present invention can remainextrachromosomal or can integrate into one or more sites within achromosome of the transformed (i.e., recombinant) cell in such a mannerthat their ability to be expressed is retained.

[0049] Suitable host cells include any cell that can be transformed witha nucleic acid molecule of the present invention, but are preferably ahost cell from an organism to which an expressed CD36 binding/antigenchimeric polypeptide will be administered. Many such cells are availableto the skilled artisan including, for example, primary cells such asfibroblasts or dendritic cells, Vero cells, non-tumorigenic mousemyoblast G8 cells (e.g., ATCC CRL 1246), K562 erythroleukemia cells,mouse NIH/3T3 cells, other fibroblast cell lines (e.g., human, murine,or chicken embryo fibroblast cell lines), myeloma cell lines, Chinesehamster ovary cells, LMTK31 cells, and/or HeLa cells. In one embodiment,the recombinant cell line is a myeloma cell line employingimmunoglobulin promoters operatively linked to the chimericpolynucleotides of the current invention.

[0050] A recombinant cell of the current invention is preferablyproduced by transforming a host cell with one or more recombinantmolecules, each comprising one or more chimeric polynucleotides of thecurrent invention operatively linked to an expression vector containingone or more transcription control sequences, examples of which aredisclosed herein. A recombinant cell of the present invention includesany cell transformed with at least one of any chimeric polynucleotide ofthe present invention. In a preferred embodiment of this aspect of theinvention, the transformed host cells are immortalized mammalian celllines capable of expressing high levels of CD36 binding/antigen chimericpolypeptides.

[0051] Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of chimeric polynucleotides of thepresent invention include, but are not limited to, operatively linkingthe polynucleotides to high-copy number plasmids, integration of thepolynucleotides into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant protein productionduring fermentation.

[0052] In another aspect, the current invention provides a method forproducing a CD36 binding/antigen fusion polypeptide in a host cell. Forthis method, an isolated chimeric CD36 binding/antigen polynucleotide isintroduced into an expression vector to produce a CD36 binding/antigenexpression vector. The CD36 binding/antigen expression vector is thenintroduced into a host cell to produce a transformed host cell. Thetransformed host cell is then maintained under conditions suitable forthe expression of CD36 binding/antigen chimeric polypeptide. Finally,the fusion polypeptide is collected.

[0053] Preferred host cells are described above. Effective cultureconditions include, but are not limited to, effective media, bioreactor,temperature, pH, and oxygen conditions that permit protein production.An effective medium refers to any medium in which a cell is cultured toproduce a fusion polypeptide of the present invention. Such mediumtypically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Cells of the present inventioncan be cultured in conventional fermentation bioreactors, shake flasks,test tubes, microtiter dishes, and petri plates. Culturing can becarried out at a temperature, pH, and oxygen conditions appropriate fora recombinant cell. Such culturing conditions are within the expertiseof one of ordinary skill in the art. Depending on the vector and hostsystem used for production, resultant polypeptides of the presentinvention can remain within the recombinant cell, be secreted into thefermentation medium or into a space between two cellular membranes(e.g., the periplasmic space in E. coli), or be retained on the outersurface of a cell or viral membrane.

[0054] The phrase “collecting the peptide”, as well as similar phrases,refers to collecting the whole medium containing the fusion polypeptideand need not imply additional steps of separation or purification.Fusion polypeptides of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing, and differential solubilization.Fusion polypeptides of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

[0055] The chimeric nucleic acids and polypeptides of the presentinvention may be administered to a host alone or in combination withanother agent. In certain embodiments, the chimeric nucleic acidmolecule or polypeptide is administered in a pharmaceutically acceptablecarrier or diluent. Many pharmaceutically-acceptable carriers anddiluents as well as methods for determining the route and quantities foradministration are known in the art.

[0056] The term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The terms “carrier” and“carrier or diluent” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Carriers are wellknown in the art. For example, saline solutions and aqueous dextrose andglycerol solutions can be employed as liquid carriers, particularly forinjectable solutions. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0057] In embodiments of the current invention wherein a polynucleotideis administered to an animal, certain preferred formulations are thoseused in NAVAC applications. The polynucleotide may be used in anaked/free form, free of any delivery vehicles. When administered in anaked/free form the polynucleotide can be simply diluted in aphysiologically acceptable solution (such as sterile saline or sterilebuffered saline) with or without a carrier. When present, the carrierpreferably is isotonic, hypotonic, or weakly hypertonic, and has arelatively low ionic strength (i.e., a sucrose solution containing, forexample, 20% sucrose).

[0058] Alternatively, the polynucleotide can be associated with agentsthat assist in cellular uptake (i.e., delivery vehicles). Deliveryvehicles include, but are not limited to, anionic liposomes,polynucleotide and/or non-polynucleotide vector components, cationiclipids, microparticles, (e.g., gold microparticles), precipitatingagents (e.g., calcium phosphate)) or any other transfection-facilitatingagent.

[0059] In certain preferred embodiments, an immunogenic composition ofthe current invention is co-administered with an adjuvant. Typically,adjuvants used with the current invention are non-toxic and do not causeundesirable side effects. Examples of adjuvants that can be used withthe current invention include, but are not limited to, aluminumhydroxide and aluminum phosphate, collectively commonly referred to asalum.

[0060] It is preferred that the chimeric nucleic acid or polypeptideinduces or enhances the immune response of the host against the antigen.To accomplish this, an effective amount of an immunogenic compositioncomprising a chimera is administered to the host. Standard techniquesmay be used to determine an effective amount of the chimera to beadministered. In particular, an effective amount may be determined bytechniques well-known to those skilled in the medical or veterinary artstaking into consideration such factors as the immunogenicity of theantigen, the condition of the animal intended for administration (i.e.,the weight, age, and general health of the animal), the mode ofadministration, and the type of formulation. The amount of immunogeniccomposition as well as a dosage regime may be adjusted to provide theoptimum induction of an immune response. In preferred embodiments, thehost is a mammal, most preferably a human.

[0061] Suitable routes of administration are many, as is known in theart. Such routes include, for example, mucosal (e.g., ocular,intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, orurinary tract), parenteral (e.g., subcutaneous, intradermal,intramuscular, intravenous, or intraperitoneal), or intranodal routes.The administration may be by injection, oral administration, inhalation,transdermal application, rectal administration, or any other route ofimmunization that enables the modulation of an animal's immune system.In certain preferred embodiments, the administration is by injection.Certain preferred routes are those that are effective for NAVACapplications, such as intramuscular, and most preferably skin. Theadministration can be achieved in a single dose or repeated atintervals.

[0062] A particularly preferred method of administering the immunogeniccompositions of the current invention is by a prime-boost protocol.Typically, an initial administration of a chimeric polynucleotide orpolypeptide composition followed by a boost with the same immunogeniccomposition, will elicit an enhanced immune response (See e.g., WO98/58956). Timing of the booster following the prime may be determinedby a skilled artisan to provide optimum response.

[0063] The following examples describe and illustrate the methods andcompositions of the invention. These examples are intended to be merelyillustrative of the present invention, and not limiting thereof ineither scope or spirit. Unless indicated otherwise, all percentages andratios are by weight. Those skilled in the art will readily understandthat variations of the materials, conditions, and processes described inthese examples can be used.

EXAMPLE 1 Preparation of Polynucleotide Constructs

[0064] This Example describes the preparation of a DNA constructencoding a chimeric polypeptide wherein the mature gp10 form of HIV-1env is ligated to two CD36 binding domains of thrombospondin, and theinsertion of the construct into an expression vector. The sequence forTSP1 was obtained from the National Center for Biotechnology Information(NCBI) (Genbank Accession # X14787; FIGS. 1B and 1C). As previouslydescribed, the TSP1—CD36 binding domains have been clearly defined astype 1 properdin-like repeats defined by the amino acid sequence CSVTCG.The antigen used in this Example is HIV gp120(mn) (Goa, F. et al., AidsRes. and Human Retroviruses, 10: 1359 (1994).

[0065] The chimeric poypeptide was constructed such that the TSP1endoplasmic reticulum targeting signal and CD36 binding domain(s) werepositioned at the NH₂-terminal end, such that the secreted polypeptidewould retain the CD36 binding domains. Because the TSP1-CD36 bindingdomains are small (i.e., 6 amino acids) and the interaction with CD36 isbelieved to be conformation dependent, the binding domains wereengineered to be expressed as in native TSP1 to retain proximalstructural conformation.

[0066] The coding sequences for the engineered portion of the fusionprotein were generated using overlapping oligonucleotides synthesized byOperon Technologies (Alameda, Calif.). The amino acid sequences of humanTSP1 that were incorporated into the chimeric construct are shown below(numbers refer to amino acids in human TSP1):

[0067] TSP1 1-31: coding sequences for signal sequence

[0068] TSP1 32-44: coding sequences for signal peptidase cleavage

[0069] TSP1 447-452: coding sequences generated CD36 Binding Domain 1

[0070] TSP1 453-463: coding sequences for a Beta Sheet region

[0071] TSP1 504-511: CD36 Binding Domain 2

[0072] The joining Beta Sheet region between the CD36 binding domains ofthrombospondin was retained in the construct because protein sequenceanalysis revealed both TSP1-CD36 domains were contained in regions ofstrong Beta Sheet conformation. To maintain conformational integrity,the spacer region engineered between Domain 1 and 2 also contained BetaSheet conformation.

[0073] The TSP1 portion of the hybrid molecule was synthesized byoverlapping and amplifying the oligonucleotides shown below: HTHROM1AATCATCCTGCAGATGGGGCTGGCCTGGGGACTAGGCGTCCTGTTCCTGATGCATGTGTGTGGCACCAACCGCATTCCAGAG HTHROM2AGACCCCTTGCGGGCGGCCCCGGTGAGTTCAAAGATGTCAAACACGCTGTTGTCTCCGCCAGACTCTGGAATGCGGTTGGTGC HTHROM3GGGGCCGCCCGCAAGGGGTCTTCTTGTTCTGTGACATGTGGTGATGGTGTGATCACAAGGATCCGGCTCTGCAAC HTHROM4ATCATCGGTACCCCATAATAGACTGTGACCCACAATTTTTCGCTCCCTCCTCCACAGGTGACAGAACAGTTGCAGAGCCGGATCCTTG

[0074] Primers HTHROM1A and HTHROM2 overlap one another and primerHTHROM3 overlaps HTHROM2 and HTHROM4. Primer HTHROM3 overlaps primerHTHROM2 and HTHROM4. To generate the full coding sequence of the TSP1portion of the hybrid, the primers were subjected to three minutes at95° C. (denaturation), three minutes at 50° C. (annealing) and 10minutes at 72° C. (extension). The resultant fragment representing thefull-length product was gel purified and further purified by PCR usingprimers HTHROM1A and HTRHOM4 for 26 cycles at 94° C. for 30 secondsfollowed by 72° C. for 60 seconds. The resulting fragment was cloned andconfirmed to be correct by DNA sequencing.

[0075] Primer HTHROM1A contains a Pst I restriction site (CTGCAG) andprimer HTHROM4 contains an Asp718 restriction site (GGTACC). These wereused to position the TSP1 portion of the chimera upstream and in-framewith the HIV gp120 portion of the hybrid (the antigen) as shown below.

[0076] The HIV gp120 coding sequences used for the engineered fusionwere assessed using published methods to identify the ER targetingsignal sequence so it could be eliminated from the sequences used toexpress gp120 in the final fusion construct (Goa et al., 1994). Signalsequences in gp120 were identified as amino acids 1-21. These aminoacids were not included in the chimeric construct. For fusions whereCD36 binding domains are upstream of the antigen of interest, it isessential to remove all intervening cleavage sequences (i.e., signalpeptidase sequences) between the CD36 binding domains and the antigen ofinterest.

[0077] For cloning purposes, the TSP1 portion of the hybrid wasmanipulated using the Pst I and Asp718 restriction sites. This 250 bpPst-I Asp718 fragment (SEQ ID NO:3) containing the engineered CD36binding domains, including sequences encoding 13 amino acids of the5′end of gp120 (up to Asp718 restriction site), was directionally clonedusing the Pst I/Asp718 sites into a prepared eukaryotic expressionvector for NAVAC.

[0078] The engineered fragment was inserted into the expression vectorin the proper orientation and in-frame with the Asp718 restriction sitein the gp120 sequences. This resulted in a continuous reading frame fromthe TSP1 signal through the CD36 binding domains terminating at thegp120 3′ carboxy terminus. The sequence was confirmed by DNA sequencingto ensure retention of the appropriate reading frame. The construct wasdesignated thrombo=gp120 (FIGS. 2A and 2B). Large quantities of purifiedthrombo=gp120 were prepared using Qiagen Giga Preparations (endotoxinfree) as per manufacturer instructions for NAVAC (Qiagen Inc. Valencia,Calif.).

EXAMPLE 2 Analysis of Immunogenicity

[0079] This Example provides an analysis of the immunogenicity ofthrombo=gp120 in mice. Mice were immunized once by an intramuscular(i.m.) route with 100 ug of DNA plasmid thrombo=gp120 expressing theTSP1-CD36 binding domain-gp120 fusion, plasmid vical gp120 expressingonly gp120, or control plasmid in 0.1 ml of PBS. As a positive controlmice were immunized with a recombinant poxvirus vP1008 (NYVAC-env; U.S.Pat. No. 5,494,807). Mice received two immunizations three weeks apart.

[0080] Both cell-mediated immunity (i.e. cytotoxic T-Cell (CTL)activity) and humoral antibody responses were analyzed in the study. ForCTL activity, pooled spleen cells of three mice from each experimentalgroup were restimulated in vitro with naive syngeneic spleen cellsinfected with vp1008 (HIV MN env). After 6 days, the spleen cells wereassayed against P815 target cells pulsed with V3 epitope peptide(peptide 121). Specific cytotoxicity was calculated as the difference incytotoxicity between peptide-pulsed P815 targets and P815 targets withno peptide pulse. Data for the CTL analysis is expressed as percentspecific lysis (i.e. difference between HIV epitope peptide pulsed andun-pulsed targets) at various effector:Target (E:T) ratios. Thisanalysis of CTL utiized Chromium-51 release assays (reviewed in Brunner,et al., “Quantitative assay of lytic action of immune cells on Cr-51labeled allogenic target cells in vivo; inhibition by isoantibody and bydrugs,” Immunology, 14:181 (1968)).

[0081] Humoral antibody responses were analyzed by bleeding three micefrom each group at the appropriate time point. Serum samples werecollected at appropriate timepoints from representative mice of bothtest and control groups, using a retroorbital plexis procedure (McGuill,M. W. and Rowan A. N. Biological Effects of Blood Loss: implications forsampling volumes and techniques. I.L.A.R. News 31: 4 (1989)) The initialbleed (week 0) was prior to the initial immunization and the (week 3)bleed was just prior the second immunization. Individual sera from eachmouse was diluted 1:400 and assayed by kinetics ELISA for antibodiesagainst recombinant HIV MN/BRU envelope glycoprotein (PMS-France) (Cox,et al., J. Natl. Cancer Inst. 71:973 (1983); and Holbrook, et al. CancerRes. 43:4019 (1983)).

[0082]FIGS. 3 and 4 summarize results of CTL analysis for thisexperiment. Three weeks after the initial immunization, CTL activity wasnot detected in mice immunized with control plasmid containing no gp120sequences or vical gp120. In contrast, strong CTL responses weregenerated by mice immunized with DNA plasmid thrombo=gp120 (FIG. 3). TheCTL responses elicited by this plasmid exceeded CTL responses generatedby an optimal dose of vP1008 (NYVAC-env) given by the optimalintraperitoneal route. Three weeks after the second immunization, miceimmunized with plasmid gp120 still had no detectable CTL activity (FIG.4). Mice immunized with thrombo=gp120 maintained the strong CTL responseseen after the first immunization.

[0083] Analysis of antibody response induced by the experimental samplesconfirmed that thrombo=gp120 constructs were effective at eliciting animmune response against gp120. Anti-HIV envelope glycoprotein antibodyassays were performed on samples collected at 0, 3, 4, and 7 weeks afterthe start of the experiment (i.e. time of initial immunization). Thestrongest, most reliable responses were generated by mice immunized withthrombo-gp120 and recombinant poxvirus vp1008 (FIG. 5). In fact, noantibody responses were detected in mice after one immunization withcontrol plasmid gp120. However, all three mice immunized one time onlywith thrombo=gp120 generated an antibody response. Antibody levels weresimilar to levels generated after immunization one time with vP1008.

EXAMPLE 3 Further Analysis of Immunogenicity

[0084] This Example provides an analysis of the immunogenicity ofthrombo=gp120 in mice that is a repeat of the analysis of Example 2carried out for longer time periods. Immunizations and CTL and antibodyassays were performed as described in Example 2. For this experiment,CTL analysis was performed at both 3 weeks and 7 weeks after the secondimmunization.

[0085] Three weeks after the second immunization, mice immunized withplasmid vical gp120 had no CTL activity above control levels (FIG. 6,Table I). However, mice immunized with thrombo=gp120 generated excellentCTL activity comparable to that generated with vP1008. Seven weeks afterthe second immunization mice immunized with vical gp120 showed a weekCTL response (FIG. 7, Table II). However, mice immunized withthrombo=gp120 showed a strong CTL response. TABLE I Specific Lysis (CTL)three weeks after primary immunization TEST ARTICLE E:T % SPECIFIC LYSISNegative control plasmid 40:1 −5.4 Negative control plasmid 20:1 −0.6Negative control plasmid 10:1 0.5 Negative control plasmid  5:1 −0.7Gp120 40:1 −1.0 Gp120 20:1 2.3 Gp120 10:1 1.2 Gp120  5:1 1.4 Thromb =gp120O 40:1 8.1 Thromb = gp120 20:1 13.1 Thromb = gp120 10:1 10.0 Thromb= gp120  5:1 0.7 VP1008 positive control 40:1 17.7 VP1008 positivecontrol 20:1 10.5 VP1008 positive control 10:1 4.1 VP1008 positivecontrol  5:1 1.5

[0086] TABLE II Specific CTL lysis, three and seven weeks after thesecond immunization TEST ARTICLE E:T % LYSIS WK 3 % LYSIS WK 7 Negativecontrol plasmid 40:1 8.9 0 Negative control plasmid 20:1 1.0 3.0Negative control plasmid 10:1 −0.1 2.6 Negative control plasmid  5:1−0.6 0.1 gp120 40:1 9.0 12.4 gp120 20:1 4.1 4.6 gp120 10:1 5.9 5.3 gp120 5:1 1.6 4.1 Thromb = gp120 40:1 59.1 58.4 Thromb = gp120 20:1 53.3 47.6Thromb = gp120 10:1 44.6 38.8 Thromb = gp120  5:1 31.9 28.5 vP1008positive control 40:1 40.3 64.4 vP1008 positive control 20:1 26.3 64.4vP1008 positive control 10:1 15.7 40.2 vP1008 positive control  5:1 7.627.5

[0087] Antibody measurements confirmed that thrombo=gp120 induced astrong immunological response against gp120. All three mice immunizedwith thrombo=gp120 generated a strong antibody response after only oneimmunization (FIG. 8). In fact, when averages of three mice for eachexperimental group were calculated, highest titers of anti-gp120antibodies were obtained following the initial immunization with eitherDNA plasmid thrombo=gp120 or the recombinant poxvirus vector pV1008(Table III).

[0088] Antibody responses following the second immunization were alsoevaluated (week 4, 6, 8, 10 of FIG. 8 and Table III). The strongantibody response of mice immunized with plasmid thrombo=gp120 continuedto escalate after the second immunization and continued throughout thestudy to show a stronger antibody titer than positive control vP1008.TABLE III Kinetics ELISA (mOD/min) WEEK Test article 0 3 4 6 8 10Negative control 1 1 1.6 0.6 1 1 gp120 1 3 22.7 29.3 27 24 Thromb =gp120 1.7 16 36.3 46.3 50 52.6 vP1008 1 8 21.6 29 31 29

[0089] The results of Examples 2 and 3 demonstrate that significantenhancement of both a cell-mediated and humoral immune response againstHIV gp120, is obtained by coupling HIV gp120 with the TSP1-CD36 bindingdomain. Not to be limited by theory, it is believed that these resultsare related to the proposed mode of immunological enhancement, (i.e.,APC Targeting), as other similar constructions expressing the identicalversion of gp120 as a fusion with other various targeting domains havefailed to enhance responses. These results suggest that the resultantimmunological enhancement is not simply associated with non-specificeffects of altered expression and or persistence (i.e., half-life) ofgp120 expressed as a fusion product.

[0090] This method of enhancing immune responses by targeting theantigen of interest to APC's can be applied to virtually anyimmunological target of interest, including those important for bothinfectious, and neoplastic diseases.

[0091] Throughout this application, various patents, publications,books, and nucleic acid and amino acid sequences have been cited. Theentireties of each of these patents, publications, books, and sequencesare hereby incorporated by reference into this application.

1 10 1 11 PRT Homo sapiens 1 Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn1 5 10 2 81 DNA Homo sapiens 2 atcatcctgc agatggggct ggcctggggactaggcgtcc tgttcctgat gcatgtgtgt 60 ggcaccaacc gcattccaga g 81 3 83 DNAHomo sapiens 3 agaccccttg cgggcggccc cggtgagttc aaagatgtca aacacgctgttgtctccgcc 60 agactctgga atgcggttgg tgc 83 4 75 DNA Homo sapiens 4ggggccgccc gcaaggggtc ttcttgttct gtgacatgtg gtgatggtgt gatcacaagg 60atccggctct gcaac 75 5 88 DNA Homo sapiens 5 atcatcggta ccccataatagactgtgacc cacaattttt cgctccctcc tccacaggtg 60 acagaacagt tgcagagccggatccttg 88 6 5722 DNA Homo sapiens 6 ggacgcacag gcattccccg cgcccctccagccctcgccg ccctcgccac cgctcccggc 60 cgccgcgctc cggtacacac aggatccctgctgggcacca acagctccac catggggctg 120 gcctggggac taggcgtcct gttcctgatgcatgtgtgtg gcaccaaccg cattccagag 180 tctggcggag acaacagcgt gtttgacatctttgaactca ccggggccgc ccgcaagggg 240 tctgggcgcc gactggtgaa gggccccgacccttccagcc cagctttccg catcgaggat 300 gccaacctga tcccccctgt gcctgatgacaagttccaag acctggtgga tgctgtgcgg 360 gcagaaaagg gtttcctcct tctggcatccctgaggcaga tgaagaagac ccggggcacg 420 ctgctggccc tggagcggaa agaccactctggccaggtct tcagcgtggt gtccaatggc 480 aaggcgggca ccctggacct cagcctgaccgtccaaggaa agcagcacgt ggtgtctgtg 540 gaagaagctc tcctggcaac cggccagtggaagagcatca ccctgtttgt gcaggaagac 600 agggcccagc tgtacatcga ctgtgaaaagatggagaatg ctgagttgga cgtccccatc 660 caaagcgtct tcaccagaga cctggccagcatcgccagac tccgcatcgc aaaggggggc 720 gtcaatgaca atttccaggg ggtgctgcagaatgtgaggt ttgtctttgg aaccacacca 780 gaagacatcc tcaggaacaa aggctgctccagctctacca gtgtcctcct cacccttgac 840 aacaacgtgg tgaatggttc cagccctgccatccgcacta actacattgg ccacaagaca 900 aaggacttgc aagccatctg cggcatctcctgtgatgagc tgtccagcat ggtcctggaa 960 ctcaggggcc tgcgcaccat tgtgaccacgctgcaggaca gcatccgcaa agtgactgaa 1020 gagaacaaag agttggccaa tgagctgaggcggcctcccc tatgctatca caacggagtt 1080 cagtacagaa ataacgagga atggactgttgatagctgca ctgagtgtca ctgtcagaac 1140 tcagttacca tctgcaaaaa ggtgtcctgccccatcatgc cctgctccaa tgccacagtt 1200 cctgatggag aatgctgtcc tcgctgttggcccagcgact ctgcggacga tggctggtct 1260 ccatggtccg agtggacctc ctgttctacgagctgtggca atggaattca gcagcgcggc 1320 cgctcctgcg atagcctcaa caaccgatgtgagggctcct cggtccagac acggacctgc 1380 cacattcagg agtgtgacaa aagatttaaacaggatggtg gctggagcca ctggtccccg 1440 tggtcatctt gttctgtgac atgtggtgatggtgtgatca caaggatccg gctctgcaac 1500 tctcccagcc cccagatgaa tgggaaaccctgtgaaggcg aagcgcggga gaccaaagcc 1560 tgcaagaaag acgcctgccc catcaatggaggctggggtc cttggtcacc atgggacatc 1620 tgttctgtca cctgtggagg aggggtacagaaacgtagtc gtctctgcaa caaccccgca 1680 ccccagtttg gaggcaagga ctgcgttggtgatgtaacag aaaaccagat ctgcaacaag 1740 caggactgtc caattgatgg atgcctgtccaatccctgct ttgccggcgt gaagtgtact 1800 agctaccctg atggcagctg gaaatgtggtgcttgtcccc ctggttacag tggaaatggc 1860 atccagtgca cagatgttga tgagtgcaaagaagtgcctg atgcctgctt caaccacaat 1920 ggagagcacc ggtgtgagaa cacggaccccggctacaact gcctgccctg ccccccacgc 1980 ttcaccggct cacagccctt cggccagggtgtcgaacatg ccacggccaa caaacaggtg 2040 tgcaagcccc gtaacccctg cacggatgggacccacgact gcaacaagaa cgccaagtgc 2100 aactacctgg gccactatag cgaccccatgtaccgctgcg agtgcaagcc tggctacgct 2160 ggcaatggca tcatctgcgg ggaggacacagacctggatg gctggcccaa tgagaacctg 2220 gtgtgcgtgg ccaatgcgac ttaccactgcaaaaaggata attgccccaa ccttcccaac 2280 tcagggcagg aagactatga caaggatggaattggtgatg cctgtgatga tgacgatgac 2340 aatgataaaa ttccagatga cagggacaactgtccattcc attacaaccc agctcagtat 2400 gactatgaca gagatgatgt gggagaccgctgtgacaact gtccctacaa ccacaaccca 2460 gatcaggcag acacagacaa caatggggaaggagacgcct gtgctgcaga cattgatgga 2520 gacggtatcc tcaatgaacg ggacaactgccagtacgtct acaatgtgga ccagagagac 2580 actgatatgg atggggttgg agatcagtgtgacaattgcc ccttggaaca caatccggat 2640 cagctggact ctgactcaga ccgcattggagatacctgtg acaacaatca ggatattgat 2700 gaagatggcc accagaacaa tctggacaactgtccctatg tgcccaatgc caaccaggct 2760 gaccatgaca aagatggcaa gggagatgcctgtgaccacg atgatgacaa cgatggcatt 2820 cctgatgaca aggacaactg cagactcgtgcccaatcccg accagaagga ctctgacggc 2880 gatggtcgag gtgatgcctg caaagatgattttgaccatg acagtgtgcc agacatcgat 2940 gacatctgtc ctgagaatgt tgacatcagtgagaccgatt tccgccgatt ccagatgatt 3000 cctctggacc ccaaagggac atcccaaaatgaccctaact gggttgtacg ccatcagggt 3060 aaagaactcg tccagactgt caactgtgatcctggactcg ctgtaggtta tgatgagttt 3120 aatgctgtgg acttcagtgg caccttcttcatcaacaccg aaagggacga tgactatgct 3180 ggatttgtct ttggctacca gtccagcagccgcttttatg ttgtgatgtg gaagcaagtc 3240 acccagtcct actgggacac caaccccacgagggctcagg gatactcggg cctttctgtg 3300 aaagttgtaa actccaccac agggcctggcgagcacctgc ggaacgccct gtggcacaca 3360 ggaaacaccc ctggccaggt gcgcaccctgtggcatgacc ctcgtcacat aggctggaaa 3420 gatttcaccg cctacagatg gcgtctcagccacaggccaa agacgggttt cattagagtg 3480 gtgatgtatg aagggaagaa aatcatggctgactcaggac ccatctatga taaaacctat 3540 gctggtggta gactagggtt gtttgtcttctctcaagaaa tggtgttctt ctctgacctg 3600 aaatacgaat gtagagatcc ctaatcatcaaattgttgat tgaaagactg atcataaacc 3660 aatgctggta ttgcaccttc tggaactatgggcttgagaa aacccccagg atcacttctc 3720 cttggcttcc ttcttttctg tgcttgcatcagtgtggact cctagaacgt gcgacctgcc 3780 tcaagaaaat gcagttttca aaaacagactcatcagcatt cagcctccaa tgaataagac 3840 atcttccaag catataaaca attgctttggtttccttttg aaaaagcatc tacttgcttc 3900 agttgggaag gtgcccattc cactctgcctttgtcacaga gcagggtgct attgtgaggc 3960 catctctgag cagtggactc aaaagcattttcaggcatgt cagagaaggg aggactcact 4020 agaattagca aacaaaacca ccctgacatcctccttcagg aacacgggga gcagaggcca 4080 aagcactaag gggagggcgc atacccgagacgattgtatg aagaaaatat ggaggaactg 4140 ttacatgttc ggtactaagt cattttcaggggattgaaag actattgctg gatttcatga 4200 tgctgactgg cgttagctga ttaacccatgtaaataggca cttaaataga agcaggaaag 4260 ggagacaaag actggcttct ggacttcctccctgatcccc acccttactc atcaccttgc 4320 agtggccaga attagggaat cagaatcaaaccagtgtaag gcagtgctgg ctgccattgc 4380 ctggtcacat tgaaattggt ggcttcattctagatgtagc ttgtgcagat gtagcaggaa 4440 aataggaaaa cctaccatct cagtgagcaccagctgcctc ccaaaggagg ggcagccgtg 4500 cttatatttt tatggttaca atggcacaaaattattatca acctaactaa aacattcctt 4560 ttctcttttt tccgtaatta ctaggtagttttctaattct ctcttttgga agtatgattt 4620 ttttaaagtc tttacgatgt aaaatatttattttttactt attctggaag atctggctga 4680 aggattattc atggaacagg aagaagcgtaaagactatcc atgtcatctt tgttgagagt 4740 cttcgtgact gtaagattgt aaatacagattatttattaa ctctgttctg cctggaaatt 4800 taggcttcat acggaaagtg tttgagagcaagtagttgac atttatcagc aaatctcttg 4860 caagaacagc acaaggaaaa tcagtctaataagctgctct gccccttgtg ctcagagtgg 4920 atgttatggg attccttttt tctctgttttatcttttcaa gtggaattag ttggttatcc 4980 atttgcaaat gttttaaatt gcaaagaaagccatgaggtc ttcaatactg ttttacccca 5040 tcccttgtgc atatttccag ggagaaggaaagcatataca cttttttctt tcatttttcc 5100 aaaagagaaa aaaatgacaa aaggtgaaacttacatacaa atattacctc atttgttgtg 5160 tgactgagta aagaattttt ggatcaagcggaaagagttt aagtgtctaa caaacttaaa 5220 gctactgtag tacctaaaaa gtcagtgttgtacatagcat aaaaactctg cagagaagta 5280 ttcccaataa ggaaatagca ttgaaatgttaaatacaatt tctgaaagtt atgttttttt 5340 tctatcatct ggtataccat tgctttatttttataaatta ttttctcatt gccattggaa 5400 tagaatattc agattgtgta gatatgctatttaaataatt tatcaggaaa tactgcctgt 5460 agagttagta tttctatttt tatataatgtttgcacactg aattgaagaa ttgttggttt 5520 tttctttttt ttgttttttt tttttttttttttttttttg cttttgacct cccattttta 5580 ctatttgcca ataccttttt ctaggaatgtgctttttttt gtacacattt ttatccattt 5640 tacattctaa agcagtgtaa gttgtatattactgtttctt atgtacaagg aacaacaata 5700 aatcatatgg aaatttatat tt 5722 71169 PRT Homo sapiens 7 Met Gly Leu Ala Trp Gly Leu Gly Val Leu Phe LeuMet His Val Cys 1 5 10 15 Gly Thr Asn Arg Ile Pro Glu Ser Gly Gly AspAsn Ser Val Phe Asp 20 25 30 Ile Phe Glu Leu Thr Gly Ala Ala Arg Lys GlySer Gly Arg Arg Leu 35 40 45 Val Lys Gly Pro Asp Pro Ser Ser Pro Ala PheArg Ile Glu Asp Ala 50 55 60 Asn Leu Ile Pro Pro Val Pro Asp Asp Lys PheGln Asp Leu Val Asp 65 70 75 80 Ala Val Arg Ala Glu Lys Gly Phe Leu LeuLeu Ala Ser Leu Arg Gln 85 90 95 Met Lys Lys Thr Arg Gly Thr Leu Leu AlaLeu Glu Arg Lys Asp His 100 105 110 Ser Gly Gln Val Phe Ser Val Val SerAsn Gly Lys Ala Gly Thr Leu 115 120 125 Asp Leu Ser Leu Thr Val Gln GlyLys Gln His Val Val Ser Val Glu 130 135 140 Glu Ala Leu Leu Ala Thr GlyGln Trp Lys Ser Ile Thr Leu Phe Val 145 150 155 160 Gln Glu Asp Arg AlaGln Leu Tyr Ile Asp Cys Glu Lys Met Glu Asn 165 170 175 Ala Glu Leu AspVal Pro Ile Gln Ser Val Phe Thr Arg Asp Leu Ala 180 185 190 Ser Ile AlaArg Leu Arg Ile Ala Lys Gly Gly Val Asn Asp Asn Phe 195 200 205 Gln GlyVal Leu Gln Asn Val Arg Phe Val Phe Gly Thr Thr Pro Glu 210 215 220 AspIle Leu Arg Asn Lys Gly Cys Ser Ser Thr Ser Val Leu Leu Thr 225 230 235240 Leu Asp Asn Asn Val Val Asn Gly Ser Ser Pro Ala Ile Arg Thr Asn 245250 255 Tyr Ile Gly His Lys Thr Lys Asp Leu Gln Ala Ile Cys Gly Ile Ser260 265 270 Cys Asp Glu Leu Ser Ser Met Val Leu Glu Leu Arg Gly Leu ArgThr 275 280 285 Ile Val Thr Thr Leu Gln Asp Ser Ile Arg Lys Val Thr GluGlu Asn 290 295 300 Lys Glu Leu Ala Asn Glu Leu Arg Arg Pro Pro Leu CysTyr His Asn 305 310 315 320 Gly Val Gln Tyr Arg Asn Asn Glu Glu Trp ThrVal Asp Ser Cys Thr 325 330 335 Glu Cys His Cys Gln Asn Ser Val Thr IleCys Lys Lys Val Ser Cys 340 345 350 Pro Ile Met Pro Cys Ser Asn Ala ThrVal Pro Asp Gly Glu Cys Cys 355 360 365 Pro Arg Cys Trp Pro Ser Asp SerAla Asp Asp Gly Trp Ser Pro Trp 370 375 380 Ser Glu Trp Thr Ser Cys SerThr Ser Cys Gly Asn Gly Ile Gln Gln 385 390 395 400 Arg Gly Arg Ser CysAsp Ser Leu Asn Asn Arg Cys Glu Gly Ser Ser 405 410 415 Val Gln Thr ArgThr Cys His Ile Gln Glu Cys Asp Lys Arg Phe Lys 420 425 430 Gln Asp GlyGly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser Val 435 440 445 Thr CysGly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro 450 455 460 SerPro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu Thr 465 470 475480 Lys Ala Cys Lys Lys Asp Ala Cys Pro Ile Asn Gly Gly Trp Gly Pro 485490 495 Trp Ser Pro Trp Asp Ile Cys Ser Val Thr Cys Gly Gly Gly Val Gln500 505 510 Lys Arg Ser Arg Leu Cys Asn Asn Pro Ala Pro Gln Phe Gly GlyLys 515 520 525 Asp Cys Val Gly Asp Val Thr Glu Asn Gln Ile Cys Asn LysGln Asp 530 535 540 Cys Pro Ile Asp Gly Cys Leu Ser Asn Pro Cys Phe AlaGly Val Lys 545 550 555 560 Cys Thr Ser Tyr Pro Asp Gly Ser Trp Lys CysGly Ala Cys Pro Pro 565 570 575 Gly Tyr Ser Gly Asn Gly Ile Gln Cys ThrAsp Val Asp Glu Cys Lys 580 585 590 Glu Val Pro Asp Ala Cys Phe Asn HisAsn Gly Glu His Arg Cys Glu 595 600 605 Asn Thr Asp Pro Gly Tyr Asn CysLeu Pro Cys Pro Pro Arg Phe Thr 610 615 620 Gly Ser Gln Pro Phe Gly GlnGly Val Glu His Ala Thr Ala Asn Lys 625 630 635 640 Gln Val Cys Lys ProArg Asn Pro Cys Thr Asp Gly Thr His Asp Cys 645 650 655 Asn Lys Asn AlaLys Cys Asn Tyr Leu Gly His Tyr Ser Asp Pro Met 660 665 670 Tyr Arg CysGlu Cys Lys Pro Gly Tyr Ala Gly Asn Gly Ile Ile Cys 675 680 685 Gly GluAsp Thr Asp Leu Asp Gly Trp Pro Asn Glu Asn Leu Val Cys 690 695 700 ValAla Asn Ala Thr Tyr His Cys Lys Lys Asp Asn Cys Pro Asn Leu 705 710 715720 Pro Asn Ser Gly Gln Glu Asp Tyr Asp Lys Asp Gly Ile Gly Asp Ala 725730 735 Cys Asp Asp Asp Asp Asp Asn Asp Lys Ile Pro Asp Asp Arg Asp Asn740 745 750 Cys Pro Pro His Tyr Asn Pro Ala Gln Tyr Asp Tyr Asp Arg AspAsp 755 760 765 Val Gly Asp Arg Cys Asp Asn Cys Pro Tyr Asn His Asn ProAsp Gln 770 775 780 Ala Asp Thr Asp Asn Asn Gly Glu Gly Asp Ala Cys AlaAla Asp Ile 785 790 795 800 Asp Gly Asp Gly Ile Leu Asn Glu Arg Asp AsnCys Gln Tyr Val Tyr 805 810 815 Asn Val Asp Gln Arg Asp Thr Asp Met AspGly Val Gly Asp Gln Cys 820 825 830 Asp Asn Cys Pro Leu Glu His Asn ProAsp Gln Leu Asp Ser Asp Ser 835 840 845 Asp Arg Ile Gly Asp Thr Cys AspAsn Asn Gln Asp Ile Asp Glu Asp 850 855 860 Gly His Gln Asn Asn Leu AspAsn Cys Pro Tyr Val Pro Asn Ala Asn 865 870 875 880 Gln Ala Asp His AspLys Asp Gly Lys Gly Asp Ala Cys Asp His Asp 885 890 895 Asp Asp Asn AspGly Ile Pro Asp Asp Lys Asp Asn Cys Arg Leu Val 900 905 910 Pro Asn ProAsp Gln Lys Asp Ser Asp Gly Asp Gly Arg Gly Asp Ala 915 920 925 Cys LysAsp Asp Phe Asp His Asp Ser Val Pro Asp Ile Asp Asp Ile 930 935 940 CysPro Glu Asn Val Asp Ile Ser Glu Thr Asp Phe Arg Arg Phe Gln 945 950 955960 Met Ile Pro Leu Asp Pro Lys Gly Thr Ser Gln Asn Asp Pro Asn Trp 965970 975 Val Val Arg His Gln Gly Lys Glu Leu Val Gln Thr Val Asn Cys Asp980 985 990 Pro Gly Leu Ala Val Gly Tyr Asp Glu Phe Asn Ala Val Asp PheSer 995 1000 1005 Gly Thr Phe Phe Ile Asn Thr Glu Arg Asp Asp Asp TyrAla Gly 1010 1015 1020 Phe Val Phe Gly Tyr Gln Ser Ser Ser Arg Phe TyrVal Val Met 1025 1030 1035 Trp Lys Gln Val Thr Gln Ser Tyr Trp Asp ThrAsn Pro Thr Arg 1040 1045 1050 Ala Gln Gly Tyr Ser Gly Leu Ser Val LysVal Val Asn Ser Thr 1055 1060 1065 Thr Gly Pro Gly Glu His Leu Arg AsnAla Leu Trp His Thr Gly 1070 1075 1080 Asn Thr Pro Gly Gln Val Arg ThrLeu Trp His Asp Pro Arg His 1085 1090 1095 Ile Gly Trp Lys Asp Phe ThrAla Tyr Arg Trp Arg Leu Ser His 1100 1105 1110 Arg Pro Lys Thr Gly PheIle Arg Val Val Met Tyr Glu Gly Lys 1115 1120 1125 Lys Ile Met Ala AspSer Gly Pro Ile Tyr Asp Lys Thr Tyr Ala 1130 1135 1140 Gly Gly Arg LeuGly Leu Phe Val Phe Ser Gln Glu Met Val Phe 1145 1150 1155 Phe Ser AspLeu Lys Tyr Glu Cys Arg Asp Pro 1160 1165 8 547 DNA Homo sapiens 8agcatctgat gcacaaaata gagtggtggt tgcttctttc cacacaggta ccccataata 60cacaattttt cgctccctcc tccacaggtg acagaacagt tgcagagccg gatccttgtg 120atcacaccat caccacatgt cacagaacaa gaagacccct tgcgggcggc cccggtgagt 180tcaaagatgt caaacacgct gttgtctccg ccagactctg gaatgcggtt ggtgccacac 240acatgcatca ggaacaggac gcctagtccc caggccagcc ccatctgcag aaaagaccca 300tggaaaggaa cagtctgtta gtctgtcagc tattatgtct ggtggcgcgc gcggcagcaa 360cgagtactgc tcagactaca ctgccctcca ccgttaacag caccgcaacg ggagttacct 420ctgactctta tcagaataca acaactcaag ctgcctgcat cttcttctgc cgctgcctta 480agtcttccat ctgcgtcagc cgtgcgagcc caatcttcac gctcattttc agacacatac 540cctaccg 547 9 547 DNA Homo sapiens 9 tcgtagacta cgtgttttat ctcaccaccaacgaagaaag ctgtgtccat gactgtgacc 60 gtgttaaaaa ggggtattat aggtctccactgtcttgtca acgtctcggc ctaggaacac 120 tagtgtggta gtggtgtaca gtgtcttgttcttctgggga acgcccgccg gggccactca 180 agtttctaca gtttgtgcga caacagaggcggtctgagac cttacgccaa ccacggtgtg 240 tgtacgtagt ccttgtcctg cggatcaggggtccggtcgg ggtagacgtc ttttctgggt 300 acctttcctt gtcagacaat cagacagtcgataatacaga ccaccgcgcg cgccgtcgtt 360 gctcatgacg agtctgatgt gacgggaggtggcaattgtc gtggcgttgc cctcaatgga 420 gactgagaat agtcttatgt tgttgagttcgacggacgta gaagaagacg gcgacggaat 480 tcagaaggta gacgcagtcg gcacgctcgggttagaagtg cgagtaaaag tctgtctatg 540 ggatggc 547 10 98 PRT Homo sapiens10 Ala Asp Ser Ala Cys Phe Leu Thr Thr Thr Ala Glu Lys Trp Val Pro 1 510 15 Val Gly Tyr Tyr Val Thr Val Trp Leu Lys Glu Ser Gly Gly Gly Cys 2025 30 Thr Val Ser Cys Asn Cys Leu Arg Ile Arg Thr Ile Val Gly Asp Gly 3540 45 Cys Thr Val Ser Cys Ser Ser Gly Lys Arg Ala Ala Gly Thr Leu Glu 5055 60 Phe Ile Asp Phe Val Ser Asn Asp Gly Gly Ser Glu Pro Ile Arg Asn 6570 75 80 Thr Gly Cys Val His Met Leu Phe Leu Val Gly Leu Gly Trp Ala Leu85 90 95 Gly Met

What is claimed is:
 1. An isolated nucleic acid molecule encoding animmunogenic chimeric polypeptide, the nucleic acid molecule comprisingat least a first nucleic acid sequence and a second nucleic acidsequence, the first nucleic acid sequence encoding at least one CD36binding domain and the second nucleic acid sequence encoding at leastone immunogenic amino acid sequence, wherein introduction of the nucleicacid molecule in a cell results in expression of the immunogenicchimeric polypeptide.
 2. The isolated nucleic acid molecule of claim 1wherein the first nucleic acid sequence is positioned 5′ of said secondnucleic acid sequence.
 3. The isolated nucleic acid molecule of claim 1comprising two first nucleic acid sequences separated from one anotherby at least one nucleic acid sequence encoding a spacer amino acidsequence.
 4. The isolated nucleic acid molecule of claim 3 wherein thefirst nucleic acid sequences are positioned 5′ of said second nucleicacid sequence.
 5. The isolated nucleic acid molecule of claim 3 or 4wherein the spacer amino acid sequence has a beta sheet conformation. 6.The isolated nucleic acid molecule or claim 3 wherein the spacer aminoacid sequence is DGVITRIRLCN.
 7. The isolated nucleic acid molecule ofclaim 1 further comprising a signal nucleic acid sequence consistingessentially of an endoplasmic reticulum signal sequence and a signalpeptidase cleavage sequence.
 8. The isolated nucleic acid molecule ofclaim 7 wherein the signal nucleic acid sequence is positioned 5′ ofboth first nucleic acid sequences.
 9. The isolated nucleic acid moleculeof claim 7 wherein the signal nucleic acid sequence is derived from thehuman thrombospondin sequence.
 10. The isolated nucleic acid molecule ofclaim 1 wherein the CD36 binding domain comprises the amino acidsequence CSVTCG.
 11. The isolated nucleic acid molecule of claim 1wherein the second nucleic acid encodes an antigen of a source selectedfrom the group consisting of a bacterium, a parasite, a virus, and acancerous cell.
 12. The isolated nucleic acid molecule of claim 11wherein the second nucleic acid encodes an antigen selected from thegroup consisting of gp100, MART-1/Melan A, gp75/TRP-1, tyrosinase,NY-ESO-1, melanoma proteoglycan, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6,MAGE-12, BAGE, GAGE-1, GAGE-2, RAGE, N-acetylglucosaminyltransferase-V,p15, β-catenin, MUM-1, cyclin dependent kinase-4, p21 ras, mutated p21ras, BCR-abl, mutated BCR-abl, p53, mutated p53, p185 HER2/neu, mutatedp185 HER2/neu, epidermal growth factor receptor, mutated epidermalgrowth factor receptor, carcinoembryonic antigens/CEA, mutatedcarcinoembryonic antigen, MUC-1, EBNA-1, E7, E6, prostate specificantigen, prostate specific membrane antigen, KSA, NY-ESO-1, and NY-BR-1.13. The isolated nucleic acid molecule of claim 11 wherein secondnucleic acid sequence encodes a gp120 polypeptide or immunogenicfragment thereof.
 14. The isolated nucleic acid molecule of claim 1 or11 further comprising a transcriptional regulatory region operablylinked to at least one of the nucleic acid sequences.
 15. The isolatednucleic acid molecule of claim 14 wherein said transcriptional controlregion is the CMV promoter.
 16. An isolated nucleic acid moleculecomprising the DNA sequence illustrated in FIG. 2A.
 17. An isolatednucleic acid molecule encoding the amino acid sequence illustrated inFIG. 2B wherein methionine is the N-terminal amino acid.
 18. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim 1 or
 11. 19. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim
 12. 20. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim
 13. 21. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim
 14. 22. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim
 15. 23. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim
 16. 24. Animmunogenic composition for generating an immune response in a host, thecomposition comprising a nucleic acid molecule of claim
 17. 25. A methodfor enhancing the immune response of a host to an antigen, the methodcomprising administering to the host an immunologically effective amountof the nucleic acid molecule of claim 1 or
 11. 26. A method of claim 25wherein the host is a mammal.
 27. A method for enhancing the immuneresponse of a host to an antigen, the method comprising administering tothe host an immunologically effective amount of the nucleic acidmolecule of claim
 12. 28. A method of claim 27 wherein the host is amammal.
 29. A method for enhancing the immune response of a host to anantigen, the method comprising administering to the host animmunologically effective amount of the nucleic acid molecule of claim13.
 30. A method of claim 29 wherein the host is a mammal.
 31. A methodfor enhancing the immune response of a host to an antigen, the methodcomprising administering to the host an immunologically effective amountof the nucleic acid molecule of claim
 14. 32. A method of claim 31wherein the host is a mammal.
 33. A method for enhancing the immuneresponse of a host to an antigen, the method comprising administering tothe host an immunologically effective amount of the nucleic acidmolecule of claim
 15. 34. A method of claim 33 wherein the host is amammal.
 35. A method for enhancing the immune response of a host to anantigen, the method comprising administering to the host animmunologically effective amount of the nucleic acid molecule of claim16.
 36. A method of claim 35 wherein the host is a mammal.
 37. A methodfor enhancing the immune response of a host to an antigen, the methodcomprising administering to the host an immunologically effective amountof the nucleic acid molecule of claim
 17. 38. A method of claim 37wherein the host is a mammal.
 39. A chimeric polypeptide comprising atleast one CD36 binding domain and at least one immunogenic amino acidsequence.
 40. The chimeric polypeptide of claim 39 wherein the CD36binding domain is positioned N-terminally from the immunogenic aminoacid sequence.
 41. The chimeric polypeptide of claim 39 comprising twoCD36 binding domains, the domains being separated from one another by atleast one spacer amino acid sequence.
 42. The chimeric polypeptide ofclaim 41 wherein the spacer amino acid sequence has a beta sheetconformation.
 43. The isolated nucleic acid molecule or claim 41 whereinthe spacer amino acid sequence is DGVITRIRLCN.
 44. The chimericpolypeptide of claim 41 further comprising an endoplasmic reticulumsignal sequence and a signal peptidase cleavage sequence.
 45. Thechimeric polypeptide of claim 44 wherein the signal sequence ispositioned N-terminally from the CD36 binding domain.
 46. The chimericpolypeptide of claim 41 wherein the CD36 binding domain comprises theamino acid sequence CSVTCG.
 47. The chimeric polypeptide of claim 41wherein the immunogenic amino acid sequence is of a source selected fromthe group consisting of a bacterium, a parasite, a virus, and acancerous cell.
 48. The chimeric polypeptide of claim 47 wherein theimmunogenic amino acid sequence is derived from gp100, MART-1/Melan A,gp75/TRP-1, tyrosinase, NY-ESO-1, melanoma proteoglycan, MAGE-1, MAGE-2,MAGE-3, MAGE-4, MAGE-6, MAGE-12, BAGE, GAGE-1, GAGE-2, RAGE,N-acetylglucosaminyltransferase-V, p15, β-catenin, MUM-1, cyclindependent kinase-4, p21 ras, mutated p21 ras, BCR-abl, mutated BCR-abl,p53, mutated p53, p185 HER2/neu, mutated p185 HER2/neu, epidermal growthfactor receptor, mutated epidermal growth factor receptor,carcinoembryonic antigens/CEA, mutated carcinoembryonic antigen, MUC-1,EBNA-1, E7, E6, prostate specific antigen, prostate specific membraneantigen, KSA, NY-ESO-1, and NY-BR-1.
 49. The chimeric polypeptide ofclaim 47 wherein the immunogenic amino acid sequence is derived fromgp120 polypeptide or an immunogenic fragment thereof.
 50. A chimericpolypeptide consisting essentially of the amino acid sequenceillustrated in FIG. 2B wherein methionine is the N-terminal amino acid.51. An immunogenic composition comprising the chimeric polypeptide ofclaim 39 in a pharmaceutically acceptable carrier.
 52. An immunogeniccomposition comprising the chimeric polypeptide of claim 40 in apharmaceutically acceptable carrier.
 53. An immunogenic compositioncomprising the chimeric polypeptide of claim 41 in a pharmaceuticallyacceptable carrier.
 54. An immunogenic composition comprising thechimeric polypeptide of claim 42 in a pharmaceutically acceptablecarrier.
 55. An immunogenic composition comprising the chimericpolypeptide of claim 43 in a pharmaceutically acceptable carrier.
 56. Animmunogenic composition comprising the chimeric polypeptide of claim 44in a pharmaceutically acceptable carrier.
 57. An immunogenic compositioncomprising the chimeric polypeptide of claim 45 in a pharmaceuticallyacceptable carrier.
 58. An immunogenic composition comprising thechimeric polypeptide of claim 46 in a pharmaceutically acceptablecarrier.
 59. An immunogenic composition comprising the chimericpolypeptide of claim 47 in a pharmaceutically acceptable carrier.
 60. Animmunogenic composition comprising the chimeric polypeptide of claim 48in a pharmaceutically acceptable carrier.
 61. An immunogenic compositioncomprising the chimeric polypeptide of claim 49 in a pharmaceuticallyacceptable carrier.
 62. An immunogenic composition comprising thechimeric polypeptide of claim 50 in a pharmaceutically acceptablecarrier.
 63. A method for enhancing the immune response of a host to anantigen, the method comprising administering to the host animmunologically effective amount of the composition of claim
 51. 64. Amethod for enhancing the immune response of a host to an antigen, themethod comprising administering to the host an immunologically effectiveamount of the composition of claim
 52. 65. A method for enhancing theimmune response of a host to an antigen, the method comprisingadministering to the host an immunologically effective amount of thecomposition of claim
 53. 66. A method for enhancing the immune responseof a host to an antigen, the method comprising administering to the hostan immunologically effective amount of the composition of claim
 54. 67.A method for enhancing the immune response of a host to an antigen, themethod comprising administering to the host an immunologically effectiveamount of the composition of claim
 55. 68. A method for enhancing theimmune response of a host to an antigen, the method comprisingadministering to the host an immunologically effective amount of thecomposition of claim
 56. 69. A method for enhancing the immune responseof a host to an antigen, the method comprising administering to the hostan immunologically effective amount of the composition of claim
 57. 70.A method for enhancing the immune response of a host to an antigen, themethod comprising administering to the host an immunologically effectiveamount of the composition of claim
 58. 71. A method for enhancing theimmune response of a host to an antigen, the method comprisingadministering to the host an immunologically effective amount of thecomposition of claim
 60. 72. A method for enhancing the immune responseof a host to an antigen, the method comprising administering to the hostan immunologically effective amount of the composition of claim
 61. 73.A method for enhancing the immune response of a host to an antigen, themethod comprising administering to the host an immunologically effectiveamount of the composition of claim 62.